D-type cyclins (D1, D2 and D3) are components of the core cell cycle machinery. It was a dogma in the cell cycle field that at least one D-cyclin must be present to allow cell proliferation. D-cyclins were regarded as essential links between cell environment and the core cell cycle machinery. During the previous funding period we challenged this dogma by studying embryos and cells lacking all three D-cyclins. Very unexpectedly, we found that the overwhelming majority of embryonal cell types proliferated normally in the absence of D- cyclins. Our analyses revealed the presence of a novel, previously unanticipated, cyclin D-independent mechanism which links the extracellular environment with activation of the core cell cycle machinery. We also found that cyclin D-null cells showed greatly reduced susceptibility to oncogenic transformation by Ras and Myc. Lastly, we found that D-cyclins were essential for proliferation of embryonic hematopoietic stem cells, and cyclin D-null embryos died due to hematopoietic stem cell depletion. This work raises several fundamental questions: (1) All analyses described above were performed using cyclin D-deficient embryonal tissues and cells. What is the requirement for cyclin D function in adult animals? The embryonal lethality of cyclin D-null mice precluded us from addressing this issue. However, in the last funding period we generated conditional cyclin D1-, D2-, and D3-knockout mouse strains. We interbred these animals, and we obtained conditional cyclin D triple-knockout mice. In Aim 1, we will use these mice to by-pass the embryonal lethality and to study cyclin D function in selected compartments of adult animals. This issue is very important from a clinical standpoint, and it must be resolved before anti-cyclin D therapy in human cancer patients is entertained. (2) Several authors proposed that D-type cyclins interact with DNA-bound tissue-specific transcription factors, and modulate their activity. We recently tested this notion in vivo using ChIP-chip technique and a novel knock-in mouse strain that we generated during the last funding period. We found the presence of cyclin D1 on promoters of several genes. In the work described in Aim 2, we will follow up on these observations with detailed mechanistic analyses. (3) We previously showed that cyclin D-null cells showed greatly reduced susceptibility to oncogenic transformation by Ras and Myc. A critical, unresolved question is whether ablation of D-cyclins in already transformed cells would revert the transformed phenotype. We will address this issue in Aim 3. Also in this Aim, we will test the requirement for cyclin D function in Ras-induced mouse lung cancer model. The Specific Aims are as follows: Aim 1: To study the function of D-cyclins in adult animals; Aim 2: To study the function of D-cyclins as transcriptional regulators; Aim 3: To study the function of D-cyclins in oncogenesis. PUBLIC HEALTH RELEVANCE: Overexpression of D-cyclins is seen in a very large fraction of human cancers. For this reason, D-cyclins are considered as potential therapeutic targets. This study will address two key issues that must be resolved before anti-cyclin D therapy in human cancer patients is entertained: (1) Are D-cyclins required for normal cell proliferation and function in the adult organism? (2) Would blocking cyclin D function in oncogenically transformed cells revert the oncogenic transformation?